Human follicle-stimulating hormone exerts a stimulatory effect on spermatogenesis, testicular size, and serum inhibin levels in the gonadotropin-releasing hormone antagonist-treated nonhuman primate (Macaca fascicularis).

The role of FSH in spermatogenesis was investigated in nonhuman primates depleted of testosterone by GnRH antagonist treatment. The GnRH antagonist antide (Nal-Lys; [N-acetyl-D-2-naphthyl-Ala1,D-4-chloro-Phe2,D-pyridyl-Ala3, nicotinyl-Lys5,D-nicotinyl-Lys6,isopropyl-Lys8,D-Ala10 ]-GnRH) was used at a daily dose of 450 micrograms/kg to suppress endogeneous gonadotropin and androgen production. Four groups of five cynomolgus monkeys (Macaca fascicularis) were subjected to the following treatment throughout a 16-week period: vehicle (group 1), GnRH antagonist (group 2), and GnRH antagonist plus human FSH (Fertinorm; 2 x 15 IU/day.animal; hFSH) during weeks 0-8 (group 3) or 8-16 (group 4). Testicular biopsies were performed before and after 4, 8, and 16 weeks of treatment. The tissue was analyzed by light microscopy and flow cytometry. Serum testosterone levels were suppressed into the range of orchidectomized animals in all GnRH antagonist-treated groups. In the absence of hFSH, serum inhibin levels were also markedly lowered. Concomitant administration of hFSH attenuated the GnRH antagonist-induced reduction of testicular size, while delayed treatment with hFSH failed to restimulate testicular volume. Numbers of A-dark spermatogonia, the reserve stem cells, were not altered by any of the treatments. hFSH either fully maintained or increased the counts for A-pale spermatogonia (renewing stem cells). The development of pachytene spermatocytes and round and elongated spermatids was markedly reduced or inhibited by the GnRH antagonist within 6-18 weeks. In contrasts, hFSH maintained these cell types at about 50% of baseline for 8 weeks. After 8 weeks of GnRH antagonist administration, hFSH stimulated A-pale spermatogonia and spermatocytes 2- to 3-fold with only minor effects on spermatid numbers. By means of flow cytometry, testicular cells were quantified according to DNA content. Within 8-16 weeks of GnRH antagonist treatment the percentage of 4C (mainly primary spermatocytes), 1C (round spermatids), and 1CC cells (elongated spermatids) had fallen from 65-75% to 5-25%. hFSH completely maintained the relative number of these cells, but failed to significantly restimulate the formation of 1CC cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The response of inhibin to human chorionic gonadotrophin is decreased in senescent men compared with young men.

Inhibin and testosterone were measured in the serum of young and old men with proven fertility before and after stimulation with human chorionic gonadotrophin (hCG) in order to characterize endocrinological changes in senescence further. While there was a significant increase of both hormones in all young men, there was a decreased response of serum testosterone and an insignificant increase in inhibin in the older men. Although basal hormone levels and ejaculate parameters were not different, hCG stimulation revealed that there were decreased secretory capacities of Leydig as well as of Sertoli cells in old age.

Sustained inhibition of sperm production and inhibin secretion induced by a gonadotrophin-releasing hormone antagonist and delayed testosterone substitution in non-human primates (Macaca fascicularis).

Since the concomitant administration of a gonadotrophin-releasing hormone (GnRH) antagonist and testosterone suppresses sperm production only incompletely, the feasibility of treatment with a GnRH antagonist and delayed testosterone supplementation for sustained suppression of sperm production in a non-human primate model was investigated. Adult cynomolgus monkeys (Macaca fascicularis; five/group) received daily s.c. injections of the GnRH antagonist [N-acetyl-D-2-naphthyl-Ala1,D-4-chloro-Phe2, D-pyridyl-Ala3,nicotinyl-Lys5,D-nicotinyl-Lys6, isopropyl-Lys8,D-Ala10]-GnRH of either 450 or 900 micrograms/kg for 18 weeks. During week 6 of the GnRH antagonist treatment, all monkeys were given a single i.m. injection of 40 mg of a long-acting testosterone ester (testosterone-trans-4-n-butylcyclo-hexanecarboxylate; 20-Aet-1). Within 1 week, serum LH bioactivity was suppressed in both groups and remained low throughout the entire treatment period. Similarly, concentrations of serum testosterone declined precipitously. During week 6, substitution with testosterone restored concentrations of serum testosterone into the pretreatment range. Concentrations of serum inhibin declined within 1 week and remained suppressed during the period of treatment with the GnRH antagonist. Testicular volumes were reduced to approximately 25% of pretreatment values in both groups by week 8 and stayed in that range during the remaining period of administration of the GnRH antagonist. During the first 6 weeks of administration of the GnRH antagonist, the ejaculatory response to electrostimulation and the volume of the ejaculates diminished with time.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of gonadotrophin secretion by inhibin, testosterone and gonadotrophin-releasing hormone in pituitary cell cultures of male monkeys.

The effects of bovine inhibin, testosterone and GnRH on gonadotrophin secretion by primate pituitary cells were characterized in vitro using pituitaries from six male rhesus monkeys and one male cynomolgus monkey. The effect of inhibin on basal secretion of FSH and LH was investigated. Dose-response curves in monkeys and rats were compared. GnRH dose-response curves in the presence and absence of testosterone were also examined in monkeys. In monkey pituitary cells, testosterone at a concentration of 10(-7) M had no effect on LH or FSH secretion. Inhibin suppressed FSH secretion to 50.8% of that of controls with no effect on LH. In rats, FSH secretion was suppressed to 45.0% of that of controls with a median effective dose (ED50, 95% range) of 1.298 (1.064-1.584) U/ml, compared with 1.024 (0.7204-1.455) U/ml in monkeys. In monkey pituitary cells, LH release was stimulated 9.9-fold and FSH 3.3-fold by GnRH. Testosterone had no effect on basal or GnRH-stimulated gonadotrophin release. These results support the view that the pituitary is not the target organ for the negative feedback action of testosterone in the male. In vitro, inhibin is the major regulator of FSH secretion at the pituitary level.

Radioimmunoassay of inhibin in the serum of male monkeys.

A heterologous inhibin radioimmunoassay method to measure inhibin in serum of male cynomolgus (Macaca fascicularis) and rhesus (Macaca mulatta) monkeys has been validated using a specific antibody against bovine 31 kDa inhibin and 125I-labelled 31 kDa inhibin as tracer. A serum pool from male monkeys was used as standard. Serial dilutions of normal monkey serum showed parallel logit-log dose-response curves to purified porcine and bovine inhibin as well as to a female human serum pool. The intra-assay coefficient of variation was 4.2% (n = 10) and the interassay coefficient of variation 5.1% (n = 10). No loss of inhibin immunoactivity was noted after storage at 23 degrees C for 5 days or repeated thawing and freezing of the serum samples. Serum from castrated monkeys showed undetectable levels of inhibin. Treatment with a gonadotrophin-releasing hormone agonist for 15 weeks led to a marked suppression of peripheral serum inhibin to concentrations similar to those after hypophysectomy or pituitary stalk section.

Effects of flutamide on testicular involution induced by an antagonist of gonadotrophin-releasing hormone and on stimulation of spermatogenesis by follicle-stimulating hormone in rats.

The effects of combined treatment with an antagonist of gonadotrophin-releasing hormone (ANT) and the antiandrogen flutamide (FL) on spermatogenesis were studied in the presence and absence of exogenous follicle-stimulating hormone (FSH). After treatment for 2 weeks, the combination of ANT (RS 68439, 450-500 micrograms/kg per day, s.c.) with 10, 20 or 40 mg FL/day, s.c. was as effective as ANT plus the Leydig cell toxin ethane dimethane sulphonate (75 mg/kg per week, i.p.) in terms of reduction in weight of testes, epididymides and seminal vesicles. Thus, a daily dose of 10 mg FL/kg was sufficient to block the androgen action in the testes of ANT-treated rats. In a second experiment, rats received ANT and ANT+FL (10 mg/kg) alone or in combination with a highly purified human FSH preparation (5 or 10 iu, twice a day) for 2 weeks. FSH did not affect testosterone concentration or weight of epididymides and seminal vesicles, but ANT+FL markedly enhanced the ANT-induced reduction of testis weight, seminiferous tubule diameter and numbers of germ cells, as revealed by qualitative and quantitative analysis of testis histology. In the absence of FL, testis size and numbers of germ cells, including elongated spermatids, were increased by FSH. In the presence of FL, the effects of FSH were less pronounced with respect to the germ cells, in terms of both numbers of cells and the effective dose of FSH. Irrespective of treatment with FL, exogenous FSH increased the inhibin concentrations in serum, indicating that Sertoli cells remained responsive to FSH. From the present study it is concluded that (i) FL accelerates ANT-induced testicular involution, (ii) FSH has a role in adult spermatogenesis and (iii) the effects of FSH on advanced germ cells are influenced by androgens.

Short-term effects of oestradiol and 4-hydroxyoestradiol on gonadotrophin-releasing hormone induced luteinizing hormone secretion by rat pituitary cells in culture.

Dispersed pituitary cells from adult female rats were preincubated for different time periods (0-12 h) in the absence or presence of 10(-9)M oestradiol (E2) or 4-hydroxyoestradiol (4-OHE2). Then the media were changed and the cells incubated for 4 h with either vehicle, or E2, or 4-OHE2 and additionally with different concentrations (10(-11)-10(-7) M) of gonadotrophin-releasing hormone (GnRH). Treatment of pituitary cells with E2 for 4 h (i.e. no preincubation with E2) significantly decreased the LH-response to GnRH at concentrations greater than or equal to 10(-10) M of the decapeptide. During a transition time of approximately 10 h (i.e. in cultures preincubated with E2 or vehicle for 2, 4, 6 or 8 h and then coincubated with E2 or vehicle and GnRH for 4 h) no differences between E2- and vehicle-treated cultures were observed. After 14 and 16 h of E2-treatment (i.e. 10 or 12 h preincubation and 4 h coincubation with GnRH) the LH-responses to GnRH in these cultures were significantly higher than in the respective controls. A nearly identical reaction pattern was observed when 4-OHE2 was used instead of E2. In a second series of experiments dispersed rat pituitary cells were suspended in a carrier gel and continuously perifused with medium, using small chromatography columns. When these cells were exposed for 4 min to 10(-9) M GnRH at 60 or 48 min intervals, they reacted with reproducible pulsatile LH-discharges during at least 6 subsequent stimuli with the decapeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Follicle-stimulating hormone stimulates inhibin in the serum of male monkeys (Macaca mulatta).

Three adult rhesus monkeys were injected intramuscularly with human FSH at doses of 2, 10 or 25 IU/kg in a cross-over design with 3-week intervals between injections. On each occasion a fourth animal received saline only as control. Serum levels of exogenous FSH were monitored by a fluoroimmunoassay specific for human FSH. Serum inhibin was measured by a heterologous radioimmunoassay. Each FSH injection was followed by a rise in serum inhibin in a dose-dependent manner. The half-life of human FSH in rhesus monkeys ranged from 25.1 to 32.9 h with no significant differences between doses. The rise of inhibin occurred with a lag time of 53.3 to 61.9 h after injection of FSH, independent of the dose administered. These findings support the concept that inhibin secretion in male primates is stimulated by FSH.

Bio and immuno-activity of FSH in serum after intramuscular injection of highly purified urinary human FSH in normal men.

The pharmacokinetics of a human FSH preparation widely used in clinical practice was evaluated in healthy male volunteers whose endogenous FSH was suppressed by 19-nortestosterone injections. 9.7 h following intramuscular injection of 150 IU FSH the mean immunoreactive peak concentration over baseline was 3.8 IU/1. Injection of 450 IU FSH led to a mean immunoreactive peak level of 10.4 IU/1 at 9.8 h. Mean half-life was 24.6 h (150 IU FSH) and 32.6 h (450 IU FSH). Serum bioFSH concentrations were suppressed below the detection limit after injection of the 19-nortestosterone and rose to preinjection levels after administration of FSH. Mean bioactive peak level for the 450 IU FSH was 7.3 IU/1 FSH at 7.4 h. The half-life of bioFSH was calculated to be 13.4 h and is considerably shorter than the half-life of immunoFSH. Serum inhibin levels were suppressed by 19-nortestosterone and did not change significantly after injection of the FSH preparation, suggesting that the FSH dose was insufficient to elicit this biological response. It is concluded that the currently used dose regimen for the treatment of male hypogonadism is inadequate for maintenance of serum bioFSH levels in the normal range.

Studies on the subcellular mechanisms mediating the negative estradiol effect on GnRH-induced LH-release by rat pituitary cells in culture.

Cultured pituitary cells from adult female rats were treated for 4 or 24 h in the absence or presence of E2 (10(-9) mol/l) with increasing concentrations of the transcription inhibitor actinomycin-D or the translation inhibitor puromycin. During the last 4 h of incubation, LH release was stimulated with 5 X 10(-10) mol/l GnRH. The positive E2 effect observed after 24 h treatment with the steroid was clearly abolished by actinomycin-D at concentrations greater than or equal to 10(-10) mol/l and by puromycin at concentrations greater than or equal to 10(-5) mol/l. These findings indicate that the positive E2 effect on GnRH-induced LH release is fully dependent on intact mRNA and protein synthesis. The negative E2 effect observed after 4 h treatment with the steroid was not affected by actinomycin-D and abolished by puromycin only at concentrations greater than or equal to 10(-4) mol/l. Similar results were obtained, when cells had been treated for 4 h with actinomycin-D or puromycin before the 4 h E2 treatment started. Thus, the negative E2 effect seems to be independent of mRNA synthesis and dependent on protein synthesis to a lesser extent than the positive E2 effect. In an attempt to identify positively the subcellular mechanism via which E2 exerts its negative effect, several steps in the GnRH stimulus secretion coupling mechanism were checked whether or not they are modulated by E2. The negative effects of E2 (10(-9) mol/l) on LH release induced by GnRH (10(-10), 10(-9) mol/l) and by the activators of voltage dependent Ca2+ channels K+ (64 mmol/l) or veratridine (3.3 X 10(-5) mol/l) were comparable to those of the calcium antagonist verapamil (10(-6) mol/l). These findings supported the speculation that E2 might act on the Ca2+ channels. The LH release induced by the Ca2+ ionophores A 23 187 (10(-4) mol/l) or ionomycin (6.6 X 10(-5) mol/l), however, was also significantly reduced by 10(-9) mol/l E2, indicating that the steroid modulated a mechanism secondary to the increase of intracellular Ca2+. Also GnRH (10(-9), 10(-8) mol/l) induced accumulation of [3H]inositol phosphates was not influenced by E2 (10(-9) mol/l) treatment, though the steroid exerted a significant negative effect on the LH release by these cells, indicating that phosphatidylinositol-4,5-biphosphate breakdown is not the point of attack for the estrogen.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for a major role of inhibin in the feedback control of FSH in the male rat.

Adult rats (16-18/group) received a single intratesticular injection of 25, 100 or 400 microliters glycerol solution (7:3 in distilled water, v/v). Half of the rats in each group were given implants of testosterone, a testosterone-filled Silastic capsule (1.5 cm length) to provide serum values of testosterone within the normal range. After 1 week all animals were killed by decapitation. Serum concentrations of gonadotrophins, testosterone and immunoactive inhibin as well as testicular concentrations of testosterone and bioactive inhibin were determined. Testicular histology was studied in Paraplast-embedded tissue stained with PAS and haematoxylin-eosin. Glycerol treatment caused a dose-dependent ablation of spermatogenesis in a distinct area around the site of injection. Serum concentrations of FSH increased proportionally with increasing spermatogenic damage while serum LH and testosterone remained unaltered except with the highest glycerol dose. The rise in serum FSH was significantly correlated with serum (r = -0.70, P less than 0.001) and testicular (r = -0.66, P less than 0.001) concentrations of inhibin. A less pronounced correlation was found between LH and serum inhibin (r = 0.48). No correlation was found between the concentrations of LH and testicular inhibin or between serum concentrations of FSH and serum testosterone in the 25 and 100 microliters groups. Maintenance of low to normal serum testosterone concentrations by means of Silastic implants blocked the elevation of FSH in glycerol-treated animals but failed to affect significantly serum FSH in untreated rats. In all testosterone treated rats testicular inhibin concentrations were markedly reduced in the presence of lowered concentrations (7-14%) of testicular testosterone and unaltered serum FSH concentrations.